Thermo Fisher Scientific TaqMan Fast Advanced Master Mix User guide

Brand
Thermo Fisher Scientific
Model
TaqMan Fast Advanced Master Mix
Type
User guide
For Research Use Only. Not for use in diagnostic procedures.
TaqMan™ Fast Advanced Master Mix
USER GUIDE
For two-step real-time RT-PCR in gene expression experiments
or quantitative analysis
Catalog Numbers 4444556, 4444557, 4444558, 4444963, 4444964, 4444965
Publication Number MAN0025706
Revision A.0
Thermo Fisher Scientific Baltics UAB | V.A. Graiciuno 8, LT-02241 | Vilnius, Lithuania
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE
LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR
ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0025706
Revision Date Description
A.0 9 November 2021 Pub. No. 4444605 Rev. D was converted to Pub. No. MAN0025706
Rev. A.0, with the following changes:
•Updated storage temperatures.
•Added QuantStudio™ 6 Pro and 7 Pro Real-Time PCR Systems.
•Added information about recommended algorithms for data
analysis.
•Added instructions to dilute 60X assays, where applicable.
•Added instructions to divide 20X assays before freezing to avoid
multiple freeze‑thaw cycles.
•Added algorithm recommendations for data analysis.
•Updated polymerase activation time for TaqMan™ Gene Expression
Assays (single‑tube and TaqMan™ Array Plates).
•Updated thermal cycling conditions for TaqMan™ Gene Expression
Assays and Custom TaqMan™ Gene Expression Assays—single-
tube assays.
•Updated PCR guidelines for TaqMan™ Gene Expression Assays—
TaqMan™ Array Cards.
•Updated anneal/extend temperature for TaqMan™ Gene Expression
Assays and TaqMan™ Array Cards on the 7900HT Fast Real-Time
PCR Instrument.
•Removed ramp rates from thermal cycling conditions for TaqMan™
Gene Expression Assays—TaqMan™ Array Cards.
•Removed use of template files to set up PCR reactions for
TaqMan™ Gene Expression Assays—TaqMan™ Array Cards. Added
import of setup files and thermal cycling conditions for this format.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is
a trademark of Roche Molecular Systems, Inc., used under permission and license. Microsoft and Excel are trademarks of Microsoft
Corporation. AmpliTaq is a registered trademark of Roche Molecular Systems, Inc.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER 1 Product information .................................................. 6
Product description ............................................................. 6
Purpose of this guide ............................................................ 6
Contents and storage ............................................................ 7
Required materials not supplied .................................................. 7
Workflow ..................................................................... 11
■CHAPTER 2 RT–PCR for TaqMan™ and Custom TaqMan™ Gene
Expression Assays—single–tube assays ........................................ 12
Perform reverse transcription .................................................... 12
Perform real-time PCR .......................................................... 12
Guidelines ................................................................ 12
Before you begin .......................................................... 12
Prepare the PCR Reaction Mix .............................................. 13
Prepare the PCR reaction plate .............................................. 13
Set up a plate document or plate file ......................................... 14
Run the PCR reaction plate ................................................. 15
Analyze data .................................................................. 15
Algorithms for data analysis ................................................. 16
■CHAPTER 3 RT–PCR for TaqMan™ Gene Expression Assays—
TaqMan™ Array Plates ............................................................ 17
Perform reverse transcription .................................................... 17
Perform real-time PCR .......................................................... 17
Guidelines ................................................................ 17
Before you begin .......................................................... 17
Prepare the PCR Reaction Mix .............................................. 18
Prepare the PCR reaction plate .............................................. 18
Set up a plate document or plate file ......................................... 19
Run the PCR reaction plate ................................................. 20
Analyze data .................................................................. 20
Algorithms for data analysis ................................................. 21
TaqMan™ Fast Advanced Master Mix User Guide 3
■CHAPTER 4 RT–PCR for TaqMan™ Gene Expression Assays—
TaqMan™ Array Cards ............................................................ 22
Perform reverse transcription .................................................... 22
Perform real-time PCR .......................................................... 22
Guidelines ................................................................ 22
Recommended amount of cDNA ............................................ 23
Before you begin .......................................................... 23
Prepare the sample–specific PCR Reaction Mix ............................... 23
Prepare the TaqMan™ Array Card ............................................ 23
Set up a card document or card file .......................................... 24
Run the TaqMan™ Array Card ................................................ 24
Analyze data .................................................................. 25
Algorithms for data analysis ................................................. 25
■CHAPTER 5 RT–PCR for TaqMan™ MicroRNA Assays—single–
tube assays ........................................................................ 26
Perform reverse transcription .................................................... 26
Perform real-time PCR .......................................................... 26
Guidelines ................................................................ 26
Before you begin .......................................................... 26
Prepare the PCR Reaction Mix .............................................. 27
Prepare the PCR reaction plate .............................................. 27
Set up a plate document or experiment file ................................... 28
Run the PCR reaction plate ................................................. 29
Analyze data .................................................................. 29
Algorithms for data analysis ................................................. 30
■CHAPTER 6 RT–PCR for TaqMan™ Advanced miRNA Assays—single–
tube assays ........................................................................ 31
Perform reverse transcription .................................................... 31
Perform real-time PCR .......................................................... 31
Guidelines ................................................................ 31
Before you begin .......................................................... 32
Prepare PCR Reaction Mix .................................................. 32
Prepare the PCR reaction plate .............................................. 32
Set up a plate document or experiment file ................................... 33
Run the PCR reaction plate ................................................. 34
Analyze data .................................................................. 34
Algorithms for data analysis ................................................. 35
Contents
4TaqMan™ Fast Advanced Master Mix User Guide
■APPENDIX A Supplemental information ......................................... 36
Components of the TaqMan™ Fast Advanced Master Mix ........................... 36
AmpliTaq™ Fast DNA Polymerase ............................................ 36
Uracil-N glycosylase ....................................................... 36
dUTP .................................................................... 36
ROX™ Passive Reference dye ............................................... 36
Two–step real-time RT–PCR ..................................................... 37
About the 5' nuclease assay ..................................................... 37
TaqMan™ MGB probes ......................................................... 38
Enzyme activation time ......................................................... 39
■APPENDIX B Best practices for PCR and RT-PCR experiments .............. 40
Good laboratory practices for PCR and RT-PCR ................................... 40
Use UNG to prevent false-positive amplification ................................... 40
Detect fluorescent contaminants ................................................. 41
■APPENDIX C Safety ............................................................... 42
Chemical safety ................................................................ 43
Biological hazard safety ......................................................... 44
■APPENDIX D Documentation and support ...................................... 45
Customer and technical support ................................................. 45
Limited product warranty ........................................................ 45
Contents
TaqMan™ Fast Advanced Master Mix User Guide 5
Product information
Product description
The Applied Biosystems™ TaqMan™ Fast Advanced Master Mix enables real-time PCR in any gene
expression experiment or quantitative analysis, including the following applications:
•Pathogen detection
•Dierential gene expression analysis
•Viral load quantitation
•MicroRNA quantitation
•Microarray verification
The Master Mix can be used with any DNA target, including complementary DNA (cDNA) or genomic
DNA (gDNA). It can be used in the second step of a two-step real-time RT–PCR protocol for RNA
quantitation experiments. A cDNA template can be generated from RNA using one of our reverse
transcription kits prior to real-time PCR with the Master Mix. See details in “Required materials not
supplied” on page 7.
The Master Mix is supplied at a 2X concentration and contains the following components:
•AmpliTaq™ Fast DNA Polymerase
•Uracil-N glycosylase (UNG)
•dNTPs with dUTP
•ROX™ Reference Dye (passive reference)
•Optimized buer components
For more information about each component, see “Components of the TaqMan™ Fast Advanced Master
Mix” on page 36.
The Master Mix is optimized for use with primers and TaqMan™ probes designed according to our
guidelines.
Purpose of this guide
This document describes how to perform two-step real-time RT-PCR using TaqMan™ Fast Advanced
Master Mix with the following assays or components:
•TaqMan™ Gene Expression Assays (single-tube assays, array plates, and array cards)
–Single‑tube assays
–TaqMan™ Array Plates
–TaqMan™ Array Cards
•Custom TaqMan™ Gene Expression Assays
1
6TaqMan™ Fast Advanced Master Mix User Guide
•TaqMan™ MicroRNA Assays
•TaqMan™ Advanced miRNA Assays
This guide provides general guidelines for analyzing data. Analysis can vary between applications. For
more information about procedures and data analysis, see the documentation for your instrument.
Contents and storage
Table 1 TaqMan™ Fast Advanced Master Mix
Cat. No. Number of 20-µL reactions Amount Storage[1]
4444556 100 1 × 1 mL
2–8°C
4444557 500 1 × 5 mL
4444963 (2 × 4444557) 1,000 2 × 5 mL
4444964 (5 × 4444557) 2,500 5 × 5 mL
4444965 (10 × 4444557) 5,000 10 × 5 mL
4444558 5,000 1 × 50 mL
[1] See label for expiration date.
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Table 2 Instrument, software, equipment, plates and accessories, and consumables
Item Source
Instrument, one of the following:
QuantStudio™ 6 Pro Real-Time PCR System[1]
Contact your local sales oce.
QuantStudio™ 7 Pro Real-Time PCR System
QuantStudio™ 3 and 5 Real-Time PCR Instruments[1]
QuantStudio™ 6 Flex Real-Time PCR System[1]
QuantStudio™ 7 Flex Real-Time PCR System
QuantStudio™ 12K Flex Real–Time PCR System
StepOne™ Real-Time PCR System[2]
StepOnePlus™ Real-Time PCR System[1]
7500 Real-Time PCR System[1]
Chapter 1 Product information
Contents and storage 1
TaqMan™ Fast Advanced Master Mix User Guide 7
Table 2 Instrument, software, equipment, plates and accessories, and consumables (continued)
Item Source
7500 Fast Real-Time PCR System[1]
Contact your local sales oce.
ViiA™ 7 Real-Time PCR System
7900HT Real-Time PCR Instrument[1]
7900HT Fast Real-Time PCR Instrument
Compatible real–time PCR instruments from other suppliers may be
acceptable. Verify thermal–cycling conditions on other real–time PCR
instruments.
Software
Microsoft™ Excel™ (Optional, to create plate layout files for import) microsoft.com
Equipment
Centrifuge with plate adapter MLS
Microcentrifuge MLS
Thermal cycler, or heat block or water bath set to 95°C MLS
Adjustable pipettors MLS
Laboratory mixer (vortex or equivalent) MLS
Tubes, plates, and other consumables
Plastics consumables thermofisher.com/plastics
Pipette tips thermofisher.com/pipettetips
Disposable gloves MLS
[1] Not compatible with TaqMan™ Array Cards.
[2] Not compatible with TaqMan™ Array Plates or TaqMan™ Array Cards.
Table 3 Reagents for reverse transcription
Item Source
Reagents for reverse transcription (all assays)
TE, pH 8.0, RNase-free AM9849
(Optional) RNase inhibitor N8080119
AM2684 (Cloned; 40 U/µL)
Nuclease-Free Water (not DEPC-Treated) AM9930
Chapter 1 Product information
Required materials not supplied
1
8TaqMan™ Fast Advanced Master Mix User Guide
Table 3 Reagents for reverse transcription (continued)
Item Source
Reagents for reverse transcription (TaqMan™ Gene Expression Assays)
High-Capacity cDNA Reverse Transcription Kit 4368814
4374966 (with RNase inhibitor)
High-Capacity RNA-to-cDNA™ Kit 4387406
SuperScript™ VILO™ cDNA Synthesis Kit 11754050
SuperScript™ IV VILO™ Master Mix 11756050
Reagents for reverse transcription (TaqMan™ MicroRNA Assays)
TaqMan™ MicroRNA Reverse Transcription Kit[1] 4366596
TaqMan™ Advanced miRNA Assays
TaqMan™ Advanced miRNA cDNA Synthesis Kit[2] A28007
[1] TaqMan™ MicroRNA Assays are optimized for use with the TaqMan™ MicroRNA Reverse Transcription Kit. Assay performance cannot be
guaranteed with other reverse transcription kits.
[2] TaqMan™ Advanced miRNA Assays are optimized for use with the TaqMan™ Advanced miRNA cDNA Synthesis Kit. Assay performance
cannot be guaranteed with other reverse transcription kits.
Table 4 Assays
Item Source
TaqMan™ Assays
TaqMan™ Gene Expression Assays thermofisher.com/taqmangeneexpression
Custom TaqMan™ Gene Expression Assays thermofisher.com/taqmancustomgeneexpression
Custom TaqMan™ probes and primers[1] thermofisher.com/customprimersprobes
TaqMan™ Array Plates
96‑well Fast (0.1‑mL) TaqMan™ Array Plates and
96‑well Standard (0.2‑mL) TaqMan™ Array Plates
thermofisher.com/taqmanarrays
TaqMan™ Array Cards
TaqMan™ Array Card thermofisher.com/taqmanarrays
TaqMan™ MicroRNA Assays
TaqMan™ MicroRNA Assays thermofisher.com/taqmanmirna
Custom TaqMan™ Small RNA Assays thermofisher.com/taqmancustommirna
TaqMan™ Advanced miRNA Assays
TaqMan™ Advanced miRNA Assays thermofisher.com/advancedmirna
[1] Synthesized to your sequence and choice of quencher and reporter dyes.
Chapter 1 Product information
Required materials not supplied 1
TaqMan™ Fast Advanced Master Mix User Guide 9
Table 5 Kits and reagents for RNA isolation
Item Source
RNA isolation products thermofisher.com/rnaisolation
Supporting reagents thermofisher.com/rnaisolationreagents
Chapter 1 Product information
Required materials not supplied
1
10 TaqMan™ Fast Advanced Master Mix User Guide
Workflow
Perform reverse transcription
▼
Perform real-time PCR amplification
Prepare the PCR Reaction Mix
▼
Prepare the PCR reaction plate or card
▼
Set up a plate or card document, or experiment file
(or use the document provided with the cards or custom plates)
▼
Run the PCR reaction plate or card
▼
Analyze the data
Chapter 1 Product information
Workflow 1
TaqMan™ Fast Advanced Master Mix User Guide 11
RT–PCR for TaqMan™ and Custom
TaqMan™ Gene Expression Assays
—single–tube assays
Perform reverse transcription
Perform reverse transcription to obtain cDNA from RNA samples.
For information on reverse transcription kits, see Table 3 on page 8. For detailed guidelines and
instructions see TaqMan™ Gene Expression Assays User Guide—single-tube assays (Pub. No. 4333458).
Perform real-time PCR
Guidelines
•Store the assays frozen.
•Protect from light until use. Excessive exposure to light might aect the fluorescent probes.
•Multiple assays can be run on one reaction plate. Include no-template controls (NTCs) for each
assay.
Before you begin
Dilute 60X assays to 20X working stocks with TE, pH 8.0, RNase-free.
•Divide the solutions into smaller aliquots to minimize freeze–thaw cycles.
Note: The aliquot size depends on how many PCR reactions you will run.
•Determine the total number of PCR reactions required, including replicates, for each sample.
Include a no-template control for each assay.
Note: We recommend four replicate reactions for each assay.
•Thaw the Master Mix on ice, then mix thoroughly but gently.
•Thaw the assays on ice, then vortex and briefly centrifuge to resuspend.
•Thaw the samples on ice, then vortex and briefly centrifuge to resuspend.
2
12 TaqMan™ Fast Advanced Master Mix User Guide
Prepare the PCR Reaction Mix
1. Combine the following components in the quantities shown, multiplied by the number of reactions
required.
Add 10% overage for pipetting loss. The minimum final dilution of RT Reaction Mix in PCR
Reaction Mix is 1:15.
Component
Volume per reaction
Final concentration
384–well, 96–well
fast (0.1–mL) plates
96–well standard
(0.2–mL), 48–well
plates
TaqMan™ Fast Advanced Master
Mix (2X) 5.0 µL 10.0 µL 1X
TaqMan™ Assay (20X) 0.5 µL 1.0 µL 1X
Nuclease-free water[1] 3.5 µL 7.0 µL —
Total volume of PCR Reaction
Mix per reaction 9.0 µL 18.0 µL —
[1] Adjust the volume of nuclease–free water for a larger volume of cDNA.
2. Vortex briefly to mix.
3. Centrifuge briefly to bring the PCR Reaction Mix to the bottom of the tube.
Prepare the PCR reaction plate
1. Transfer the appropriate volume of PCR Reaction Mix to each well of the plate.
2. Add cDNA template (1 pg to 100 ng in nuclease–free water), or nuclease–free water for NTC, to
each well.
•384-well plate, 96–well fast (0.1 mL) plate: 1.0 µL
•96–well standard (0.2 mL) plate, 48–well plate: 2.0 µL
Note: Adjust the volume of nuclease–free water in the PCR Reaction Mix for a larger volume of
cDNA.
3. Seal the reaction plate with optical adhesive film, then centrifuge briefly to bring the PCR Reaction
Mix to the bottom of the well.
4. Apply a compression pad to the plate, if required by your real-time PCR system.
Chapter 2 RT–PCR for TaqMan™ and Custom TaqMan™ Gene Expression Assays—single–tube assays
Perform real-time PCR 2
TaqMan™ Fast Advanced Master Mix User Guide 13
Set up a plate document or plate file
See the appropriate instrument user guide for detailed instructions to program the thermal-cycling
conditions or to run the plate.
Note: The instrument must be configured with the block appropriate for the plate type.
1. Set up a plate document or plate file using the following conditions.
Real–time PCR system
(Optional) UNG
incubation
Polymerase
activation[1] PCR (40 cycles)
Hold
50°C
Hold
95°C
Denature
95°C
Anneal / extend
60°C
•QuantStudio™ 6 Pro and 7 Pro
Real-Time PCR Systems
• QuantStudio™ 3/QuantStudio™ 5
Flex Real‑Time PCR System
•QuantStudio™ 6 / QuantStudio™
7 Flex Real-Time PCR System
•QuantStudio™ 12K Flex Real–
Time PCR System
•StepOne™ Real-Time PCR
System
•StepOnePlus™ Real-Time PCR
System
•ViiA™ 7 Real-Time PCR System
•7900HT Real-Time PCR System
•7900HT Fast Real‑Time PCR
System
2 minutes 20 seconds[2] 1 second 20 seconds
•7500 Fast Real-Time PCR
System
•7500 Real-Time PCR System
2 minutes 20 seconds[2] 3 seconds 30 seconds
[1] To activate AmpliTaq™ Fast DNA Polymerase.
[2] Enzyme activation can continue for up to 2 minutes without affecting the results. See “Enzyme activation time” on page 39.
2. Select the appropriate block, if this option applies to your instrument.
3. Select the appropriate experiment type, if this option applies to your instrument.
4. Select TaqMan™ Reagents to detect the target sequence, if this option applies to your instrument.
Chapter 2 RT–PCR for TaqMan™ and Custom TaqMan™ Gene Expression Assays—single–tube assays
Perform real-time PCR
2
14 TaqMan™ Fast Advanced Master Mix User Guide
5. Select a run mode.
Real-time PCR system Run mode
•7500 Real-Time PCR System
•7900HT Real-Time PCR System
•7900HT Fast Real‑Time PCR System (384–well and 96–well standard
block modules)
Standard
•QuantStudio™ 6 Pro and 7 Pro Real-Time PCR Systems
•QuantStudio™ 3/QuantStudio™ 5 Flex Real‑Time PCR System
•QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time PCR System
•QuantStudio™ 12K Flex Real–Time PCR System
•StepOne™ Real-Time PCR System
•StepOnePlus™ Real-Time PCR System
•ViiA™ 7 Real-Time PCR System
•7500 Fast Real-Time PCR System
•7900HT Fast Real‑Time PCR System (384–well and 96–well standard
block modules)
Fast
6. Enter the sample volume, if this option applies to your instrument.
•384-well plate, 96–well fast (0.1 mL) plate: 10.0 µL
•96–well standard (0.2 mL) plate, 48–well plate: 20.0 µL
Run the PCR reaction plate
1. Open the plate document or experiment file that corresponds to the reaction plate in the system
software.
2. Load the reaction plate.
3. Start the run.
Analyze data
Data analysis varies depending on your real–time PCR system. See the instrument user guide for more
information.
1. View the amplification plots for the reactions.
2. Use auto baseline and auto threshold settings, or set the baseline and threshold values to
determine the threshold cycles (Ct) for the amplification curves.
3. Use the relative standard curve method or the comparative Ct method to analyze data.
Chapter 2 RT–PCR for TaqMan™ and Custom TaqMan™ Gene Expression Assays—single–tube assays
Analyze data 2
TaqMan™ Fast Advanced Master Mix User Guide 15
Algorithms for data analysis
Table 6 Algorithm recommendations for single–tube assays
Algorithm Recommendation
Threshold (Ct) Recommended.
Relative threshold (Crt)(Optional) Use for troubleshooting abnormal or unexpected results.
The relative threshold algorithm is available in the Relative Quantification application on Thermo Fisher™
Connect (thermofisher.com/connect).
Chapter 2 RT–PCR for TaqMan™ and Custom TaqMan™ Gene Expression Assays—single–tube assays
Analyze data
2
16 TaqMan™ Fast Advanced Master Mix User Guide
RT–PCR for TaqMan™ Gene
Expression Assays—TaqMan™ Array
Plates
Perform reverse transcription
Perform reverse transcription to obtain cDNA from RNA samples.
For information on reverse transcription kits, see Table 3 on page 8. For detailed guidelines
and instructions see TaqMan™ Gene Expression Assays User Guide—TaqMan™ Array Plates (Pub.
No. 4391016).
Perform real-time PCR
Guidelines
Store the plates away from light until use. Excessive exposure to light might aect the fluorescent
probes.
Before you begin
•Determine the total number of PCR reactions.
One reaction corresponds to one well in the plate.
•Thaw the Master Mix on ice, then mix thoroughly but gently.
•Thaw the samples on ice, then vortex and briefly centrifuge to resuspend.
3
TaqMan™ Fast Advanced Master Mix User Guide 17
Prepare the PCR Reaction Mix
1. Combine the following components for the number of reactions required.
Add 10% overage for pipetting loss.
Component
Volume per reaction
96–well fast (0.1–mL) plate 96–well standard (0.2–mL)
plate
cDNA template + Nuclease-free water[1] 5 µL 10 µL
TaqMan™ Fast Advanced Master Mix (2X) 5 µL 10 µL
Total volume of PCR Reaction Mix per
reaction 10 µL 20 µL
[1] 5–50 ng of cDNA diluted in nuclease–free water.
2. Vortex briefly to mix.
3. Centrifuge briefly to bring the PCR Reaction Mix to the bottom of the tube.
Prepare the PCR reaction plate
1. Transfer the appropriate volume of PCR Reaction Mix to each well of the plate.
•96–well fast (0.1 mL) plate: 10 µL
•96–well standard (0.2 mL) plate: 20 µL
Note: Adjust the volume of nuclease–free water in the PCR Reaction Mix for a larger volume of
cDNA.
2. Seal the plate with optical adhesive film, then centrifuge briefly to bring the PCR Reaction Mix to
the bottom of the wells.
3. Apply a compression pad to the plate, if required by your real-time PCR system.
Chapter 3 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array Plates
Perform real-time PCR
3
18 TaqMan™ Fast Advanced Master Mix User Guide
Set up a plate document or plate file
See the appropriate instrument user guide for detailed instructions to program the thermal-cycling
conditions or to run the plate.
Note: The instrument must be configured with the block appropriate for the plate type.
1. Import the setup file (SDS in TXT format) into the real-time PCR instrument or software.
2. Set up a plate document or plate file using the following conditions.
Real–time PCR system
(Optional) UNG
incubation
Polymerase
activation[1] PCR (40 cycles)
Hold
50°C
Hold
95°C
Denature
95°C
Anneal / extend
60°C
•QuantStudio™ 6 Pro and 7 Pro
Real-Time PCR Systems
• QuantStudio™ 3/QuantStudio™ 5
Flex Real‑Time PCR System
•QuantStudio™ 6 / QuantStudio™
7 Flex Real-Time PCR System
•QuantStudio™ 12K Flex Real–
Time PCR System
•StepOnePlus™ Real-Time PCR
System
•ViiA™ 7 Real-Time PCR System
•7900HT Real-Time PCR System
•7900HT Fast Real‑Time PCR
System
2 minutes 20 seconds[2] 1 second 20 seconds
•7500 Fast Real-Time PCR
System
•7500 Real-Time PCR System
2 minutes 20 seconds[2] 3 seconds 30 seconds
[1] To activate AmpliTaq™ Fast DNA Polymerase.
[2] Enzyme activation can continue for up to 2 minutes without affecting the results. See “Enzyme activation time” on page 39.
3. Select the appropriate block, if this option applies to your instrument.
4. Select the appropriate experiment type, if this option applies to your instrument.
5. Select TaqMan™ Reagents to detect the target sequence, if this option applies to your instrument.
Chapter 3 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array Plates
Perform real-time PCR 3
TaqMan™ Fast Advanced Master Mix User Guide 19
6. Select a run mode.
Real-time PCR system Run mode
•7500 Real-Time PCR System
•7900HT Real-Time PCR System
•7900HT Fast Real‑Time PCR System
Standard
•QuantStudio™ 6 Pro and 7 Pro Real-Time PCR Systems
•QuantStudio™ 3/QuantStudio™ 5 Flex Real‑Time PCR System
•QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time PCR System
•QuantStudio™ 12K Flex Real–Time PCR System
•StepOnePlus™ Real-Time PCR System
•ViiA™ 7 Real-Time PCR System
•7500 Fast Real-Time PCR System
•7900HT Fast Real‑Time PCR System (384–well and 96–well standard
block modules)
Fast
7. Enter the sample volume, if this option applies to your instrument.
•96–well fast (0.1 mL) plate: 10.0 µL
•96–well standard (0.2 mL) plate: 20.0 µL
Run the PCR reaction plate
1. Open the plate document or experiment file that corresponds to the reaction plate in the system
software.
2. Load the reaction plate.
3. Start the run.
Analyze data
Data analysis varies depending on your real–time PCR system. See the instrument user guide for more
information.
1. View the amplification plots for the reactions.
2. Set the baseline and threshold values to determine the threshold cycles (Ct) for the amplification
curves, or select relative threshold under analysis settings to obtain (Crt) values.
3. Use the relative standard curve method or the comparative Ct method to analyze data.
Chapter 3 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array Plates
Analyze data
3
20 TaqMan™ Fast Advanced Master Mix User Guide
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Thermo Fisher Scientific TaqMan Fast Advanced Master Mix User guide

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Thermo Fisher Scientific
Model
TaqMan Fast Advanced Master Mix
Type
User guide
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